Follicle stimulating hormone superagonists

ABSTRACT

Modified follicle stimulating hormone (FSH), containing an amino acid sequence which differs from the wild-type FSH, said modified FSH comprising a modified α-subunit, wherein the potency of said modified FSH is increased by at least about a ten fold as compared to wild type FSH. Nucleic acids encoding the modified FSH, vectors comprising the nucleic acids, and host cells comprising the vectors. Methods of assisting reproduction, diagnosing and/or treating conditions associated with glycoprotein hormone activity, reducing hyperstimulation syndrome, improving the quality of oocytes, inducing superovulation, and enhancing in vitro fertilization.

FIELD OF INVENTION

This invention relates generally to modified follicle stimulatinghormones (FSH) having superagonist activity, and the use thereof in thetreatment of conditions associated with glycoprotein hormone activity.More specifically, this invention relates to modified FSH moleculescontaining two or more amino acid substitutions as compared to wild typeFSH, wherein such modified FSH molecules exhibit enhancedpharmacological properties as compared to wild type FSH.

BACKGROUND OF INVENTION

Follitropin (follicle-stimulating hormone, FSH) and the gonadotropinschorionic gonadotropin, (CG), lutropin (luteinizing hormone, LH), andthyrotropin (thyroid-stimulating hormone, TSH) comprise the family ofglycoprotein hormones. Each hormone is a heterodimer of twonon-covalently linked subunits: alpha and beta. Within the same species,the amino acid sequence of the alpha-subunit is identical in all thehormones, whereas the sequence of the beta-subunit is hormone specific(Pierce, J. G. and Parsons, T. F. “Glycoprotein hormones: structure andfunction.” Ann. Rev. Biochem. 50:465-495 (1981)). The fact that thesequences of the subunits are highly conserved from fish to mammalsimplies that these hormones have evolved from a common ancestral protein(Fontaine Y-A. and Burzawa-Gerard, E. “Esquisse de l′evolution deshormones gonadotopes et thyreotropes des vertebres.” Gen. Comp.Endocrinol. 32:341-347 (1977)).

Recombinant follitropin has been used in certain therapies, such as inthe treatment of patients suffering from infertility (Lathi and Milki,“Recombinant gonadotropins,” Curr Womens Health Rep. 1(2):157-63(2001)). The hormone has been used in women to induce ovulation, andalso in men to induce spermatogenesis (Bouloux et al., “Induction ofspermatogenesis by recombinant follicle-stimulating hormone (puregon) inhypogonadotropic azoospermic men who failed to respond to humanchorionic gonadotropin alone,” J Androl. 24(4):604-11 (2003)), improvedisturbed sperm structures (Haidl et al., “Drug treatment of malefertility disorders,” Asian J Androl. 2(2):81-5 (2000)), and treatconditions associated with decreased levels of testosterone (see U.S.Pat. Nos. 5,574,011 and 6,562,790, each incorporated by reference). Theresponse of women to exogenous FSH therapy, has been shown to bevariable, with some demonstrating a poor response to a standard therapyprotocol (requiring adjustment of the FSH doses), and othersdemonstrating ovarian hyperstimulation syndrome (Perez et al., “Ovarianresponse to follicle-stimulating hormone (FSH) stimulation depends onthe FSH receptor genotype,” J Clin Endocrinol Metab. 85(9):3365-9(2000)). What is needed are modified derivatives of FSH having increasedactivity, to facilitate treatment of poor responders while permittinglower dose therapy regimens of patients prone to ovarianhyperstimulation.

SUMMARY OF INVENTION

This invention encompasses modified FSH proteins and nucleic acidsencoding the same, wherein the in vivo and in vitro bioactivities of themodified proteins are substantially increased as compared to wild typeFSH. In particular, the modified analogs of the invention demonstratesurprisingly enhanced pharmacological properties, including potency andVmax (efficacy), as compared to wild type FSH. Further, the modifiedanalogs of the invention provide dramatic increases in the quantity andquality of oocytes, blastocysts and embryos of treated animals. Theanalogs of the invention thus provide a long awaited solution for a widespectrum of patients suffering from infertility, including womendemonstrating a poor response following in vitro fertilization (IVF),women who have been disqualified from IVF, women demonstrating lownumbers of FSH receptors and women with FSH receptor mutations leadingto infertility.

The modified FSH molecules of the invention contain at least a modifiedα-subunit containing a combination of at least two mutations inperipheral loops of FSH, which lead to a modified FSH having increasedpotency over wild type FSH or modified proteins comprising the specifiedmutations alone. Typically, the modified FSH proteins of the inventiondemonstrate at least about a ten fold increase in potency over wild typeFSH, with preferred α-subunit mutations comprising at least two basicamino acids at positions corresponding to positions 13, 14, 16, 17, 20,21, 22, 66, 68, 73, 74 and 81 of SEQ ID No. 1.

The modified FSH proteins of the invention may further comprise amodified β-subunit, particularly a modified β-subunit comprising atleast one basic amino acid at a position corresponding to any one ofpositions 2, 4, 14, 63, 64, 67 and 69 of SEQ ID No. 2. The modified FSHproteins of the invention may also demonstrate an increased or decreasedplasma half-life as compared to wild type FSH or a decreased plasmahalf-life as compared to wild type FSH. An increase in plasma half-lifemay be facilitated by pegylation, by inclusion of a potentialglycosylation site or by other means.

The invention also includes methods of assisting reproduction in asubject comprising administering an assisting amount of the modified FSHof the invention, for instance in an in vitro fertilization protocol orartificial insemination protocol or other protocol in which ovulation orspermatogenesis is induced. Also included are methods of diagnosing andtreating conditions associated with glycoprotein hormone activity inwomen, including but not limited to ovulatory dysfunction, luteal phasedefects, time-limited conception, low FSH receptor expression in growingfollicles, low FSH receptor sensitivity, FSH receptor binding and/orcoupling deficiencies, pituitary failure or injury, unexplainedinfertility and ovarian carcinoma. The modified FSH proteins of theinvention are particularly useful for treating women prone to ovarianhyperstimulation, where analogs with the longest half-life may beapplied early in the cycle and those with shorter half-life later in thecycle to prevent or reduce the possibility of ovarian hyperstimulationsyndrome (OHHS). Also included are methods of diagnosing and treatingconditions associated with glycoprotein hormone activity in men,including but not limited to male factor infertility, pituitary failureor injury, male pattern baldness, testicular carcinoma and any conditionassociated with deficient levels of testosterone production.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-E are graphs showing a comparison of the effect of varioussingle mutations on FSH bioactivity in vitro compared to wild type (WT),as measured using transient transfection of CHO-FSHR cells.

FIG. 2 is a graph showing the effect of the beta E4R mutation on hFSHproduction in transfected CHO-FSHR cells.

FIGS. 3A and 3B are graphs showing a comparison of the effect of variouscombined mutations on FSH bioactivity in vitro, as measured usingtransient transfection of CHO-FSHR cells.

FIGS. 4A and B are graphs showing a comparison of cross-reactivity ofrat and human LHR to analog FSH and wild type FSH. FIG. 4A showscross-reactivity between FSH TR-4402 and rat luteinizing hormonereceptor. FIG. 4B shows that there is no cross-reactivity between FSHTR-4402 and human luteinizing hormone.

FIG. 5 is a diagram of the structure of FSH showing the loops in thealpha and beta subunits.

FIGS. 6A and B are graphs showing cAMP production in CHO cells inresponse to purified analog TR-4402 versus wild type FSH and purifiedanalog TR-4401 versus wild type FSH, respectively.

FIG. 7 is a graph showing cAMP production in KGN cells in response topurified analog TR-4402 versus wild type FSH.

FIG. 8 is a graph showing cAMP production in GLHR-15 cells in responseto purified analog TR-4402 versus wild type FSH.

FIG. 9 is a graph showing follicle survival in the presence of wild typehFSH (compound #3) and analog TR-4402 (compound #4), observed during anin vitro follicle bioassay.

FIG. 10 is a graph showing antrum formation in the presence of wild typehFSH (compound #3) and analog TR-4402 (compound #4), observed during anin vitro follicle bioassay.

FIG. 11A is a graph showing mucification of COC in the presence of wildtype hFSH (compound #3) and analog TR-4402 (compound #4), observedduring an in vitro follicle bioassay. FIG. 11B is a graph showing %oocyte release upon hCG stimulation in the presence of wild type hFSH(compound #3) and analog TR-4402 (compound #4), observed during an invitro follicle bioassay.

FIG. 12 is a graph showing oocyte nuclear maturation as measured by PBextrusion in the presence of wild type hFSH (compound #3) and analogTR-4402 (compound #4), observed during an in vitro follicle bioassay.

FIGS. 13A and B are graphs showing progesterone production in thepresence of wild type hFSH (compound #3) (13A) and analog TR-4402(compound #4) (13B), observed during an in vitro follicle bioassay.

FIGS. 14A-D show the results of a Steelman-Pohley Bioassay performedusing immature Sprague-Dawley Female Rats (Steelman and Pohley, 1953).The graphs in FIGS. 14A, C and D show differences in ovarian weightmeasured in response to TR-4402 as compared to wild type (Follistim).The graph in FIG. 14B compares the serum levels of TR-4402 and wild typeFSH during the bioassay.

FIG. 15 is a graph showing the intra-ovarian estradiol content of ratstreated with wild type FSH (Follistim) as compared to rats treated withthe analog TR-4402.

FIG. 16 is a graph showing serum inhibin B levels in rats afterstimulation with corresponding doses of wild type FSH (Follistim) andthe analog TR-4402.

FIGS. 17 A and B are graphs showing the elimination and absorption ofFSH analogs TR-4901, TR-4401, and TR-4402 versus wild type FSH.

FIG. 18 shows N-terminal extensions which can be used to prolong halflife of FSH analogs (SEQ. ID Nos. 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12).

FIG. 19 is a graph showing cAMP production in CHO cells in response toLA1-4402 (TR-4402 further modified to increase FSH serum half-life)versus LA1 FSH (FSH modified to increase FSH serum half-life), TR-4402,and wild type FSH.

FIG. 20 is a graph showing an increase in number of ovulated oocytesproduced in vivo in response to LA1-4402 (TR-4402 modified to increaseserum half-life) versus wild type FSH (Follistem) and hCG only.

FIG. 21 is a graph showing the increase in total number of oocytesproduced in vivo after administration of TR 4401 versus wild type FSH(Gonal F).

FIG. 22 is a graph showing the increase in fertilization rate of oocytesafter in vivo administration of TR 4401 versus wild type FSH (Gonal F).

FIG. 23 is a graph showing the increase in blastocyst formation rateafter in vivo administration of TR 4401 versus wild type FSH (Gonal F).

FIG. 24 is a graph showing the increase in total number of embryos afterin vivo administration of TR 4401 versus wild type FSH (Gonal F).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides modified “superactive” FSH moleculesshowing surprisingly enhanced potency as compared to wild type FSH.Being “modified” means that, while the protein contains an amino acidsequence which differs from the wild-type FSH, the sequence has not beenchanged such that it is identical to the known FSH sequence of anotherspecies. “Superactivity” may be assessed according to a variety ofparameters, including potency and efficacy. “Potency” is a parameter ofbioactivity that is determined by measuring the half maximal response.Differences in “potency” are determined by comparing the value of theFSH response of the analog halfway between baseline and maximum (EC50)versus that of wild type FSH. FSH responses may be measured in vitrousing purified proteins, or may be estimated following transienttransfection of a nucleic acid encoding the modified protein. FSHresponses may also be measured in vivo, i.e. in an animal responsive tosaid FSH analog. Such responses encompass any known cellular orbiological and quantitative or qualitative response of FSH binding toits receptor, i.e. cAMP production, synthesis of proteins such asprogesterone, fertilization rate, blastocyst formation rate, embryodevelopment per fertilized oocyte, etc.

“Efficacy” (Vmax) or maximum response is another parameter ofbioactivity. As discussed herein, parameters of bioactivity may varydepending on receptor number and receptor coupling in the assay cellline. In systems with lower receptor numbers or impaired coupling,differences are more discernable in terms of Vmax (efficacy). In systemswhere receptors are overexpressed, differences in potency are morevisible.

In vivo quantitative and qualitative parameters such as quantity ofoocytes, fertilization rate and blastocyst and embryo formation ratesmay be measured at the maximally effective dose for oocyte number. Themaximally effective dose for oocyte number is the optimal amount ofsuperactive FSH for both oocyte quality and quantity. The maximallyeffective dose for oocyte number is dependent on an animal's weight andrate of metabolism. For example, the maximally effective dose for alarger animal with a slower rate of metabolism is greater than themaximally effective dose for a smaller animal with a higher rate ofmetabolism. The maximally effective dose is determined empirically foreach animal.

However, regardless of the system used, the modified superactive FSHproteins of the invention demonstrate at least about a 10 fold increasein potency more preferably at least about a 20 fold, 30 fold, 40 fold,50 fold, 60 fold, 70 fold, 80 fold, 90 fold or even 100 fold increase inpotency compared to wild type FSH, or about a 10% increase in maximalefficacy, more preferably at least a 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or even 100% increase in maximal efficacy compared to wild typeFSH. The superactive analogs of the invention may also provide about afive to ten fold increase in potency or 5% to 10% increase in maximalefficacy as compared to wildtype FSH. Some of the modified proteins ofthe invention demonstrate at least about a thirty to fifty fold increasein potency or 30% to 50% increase in maximal efficacy as compared towild type. Thus, the modified FSH proteins of the present invention areparticularly useful for treating patients with low receptor number ordeficiencies in receptor response, since the modified proteins of theinvention maintain at least a 10 fold increase in potency or 10%increase in maximal efficacy even in systems with low receptor number orresponse.

The rate of absorption of a modified superactive FSH may result inincreased or decreased duration of action. A modified FSH analog with anincreased rate of absorption and decreased duration of action may bebeneficial for hypersensitive patients at risk for hyperstimulationsyndrome. The rate of absorption is measured by K_(a). The rate ofelimination is measured by K_(e).

The modified FSH molecules of the invention include modified proteins ofspecies selected from the group consisting of human, bovine, equine,porcine, ovine, murine, rat, rabbit, primate, fish, etc. Fish FSH (alsoknown as GTH-1) may be used in aquaculture, i.e., in order to growendangered or other fish species in captivity. Other species of modifiedFSH find use in agriculture breeding, and in the laboratory setting fortesting the effects of different combined mutations on various male andfemale glycoprotein hormone-related conditions. The modified FSHmolecules of other species have substitutions at positions correspondingto those in the modified human FSH molecules disclosed herein, which maybe identified using any alignment program, including but not limited toDNASIS, ALIONment, SIM and GCG programs such as Gap, BestFit, FrameAlignand Compare.

Modified human FSH molecules of the present invention comprise at leasta modified α-subunit, wherein the alpha subunit comprises at least twobasic amino acids such as those at positions corresponding to positions13, 14, 16, 17, 20, 21, 22, 66, 68, 73, 74 and 81 of wild type human FSHalpha (SEQ ID No. 1). The modified proteins may also contain a modifiedbeta subunit, wherein the beta subunit comprises at least one basicamino acid at positions corresponding to positions 2, 4, 14, 63, 64, 67and 69 of wild type human FSH beta (SEQ ID No. 2). The modified proteinsof the invention may also contain further substitutions, particularlyconservative substitutions that do not alter the enhanced properties ofthe protein. Typically, however, such modified proteins will containless than five substitutions at positions other than those listed above,and may exhibit complete amino acid sequence identity with thecorresponding wild-type FSH alpha and beta subunits in positions otherthan the positions listed above.

Basic amino acids comprise the amino acids lysine, arginine, andhistidine, and any other basic amino acid which may be a modification toany of these three amino acids, synthetic basic amino acids not normallyfound in nature, or any other amino acid which is positively charged ata neutral pH. Preferred basic amino acids, among others, are selectedfrom the group consisting of lysine and arginine.

Exemplary modified FSH molecules having two basic amino acidsubstitutions include but are not limited to proteins with substitutionsat positions 14 and 66 of the α-subunit, particularly E14R and N66R,positions 14 and 73 of the α-subunit, particularly E14R and G73Rpositions 16 and 20 of the α-subunit, particularly P16R and Q20R, andpositions 20 and 21 of the α-subunit, particularly Q20R and P21R.

The modified FSH proteins of the invention may also have an α-subunitcomprising three basic amino acid substitutions at positions selectedfrom the group consisting of positions 13, 14, 16, 17, 20, 21, 22, 66,68, 73, 74 and 81. Such modified proteins include but are not limited toproteins with combined substitutions at positions 16, 20 and 21,particularly P16R, Q20R and P21R, positions 14, 20 and 73, particularlyE14R, Q20R and G73R, positions 66, 73 and 81, particularly N66K, G73Kand A81K, positions 14, 66 and 73, particularly E14R, N66R and G73R, andpositions 14, 21 and 73, particularly E14R, P21R and G73R.

The modified FSH proteins of the invention may also have an α-subunitcomprising four basic amino acid substitutions at positions selectedfrom the group consisting of positions 13, 14, 16, 17, 20, 21, 22, 66,68, 73, 74 and 81. Such modified proteins include but are not limited toproteins with combined substitutions at positions 13, 14, 16 and 20,particularly the combination of Q13R, E14R, P16R and Q20R, and thecombination of Q13K, E14K, P16K and Q20K.

The modified FSH proteins of the invention may also have an α-subunitcomprising five basic amino acid substitutions at positions selectedfrom the group consisting of positions 13, 14, 16, 17, 20, 21, 22, 66,68, 73, 74 and 81. Such modified proteins include but are not limited toproteins with combined substitutions at positions 14, 20, 21, 66 and 73,particularly E14R, Q20R, P21R, N66R and G73R, and positions 14, 16, 20,66 and 73, particularly E14R, P16R, Q20R, N66R and G73R.

The modified FSH proteins of the invention may also have an α-subunitcomprising six basic amino acid substitutions at positions selected fromthe group consisting of positions 13, 14, 16, 17, 20, 21, 22, 66, 68,73, 74 and 81. Such modified proteins include but are not limited toproteins with combined substitutions at positions 13, 14, 16, 20, 66 and73, particularly Q13K, E14K, P16K, Q20K, N66K and G73K, and positions14, 16, 20, 21, 66 and 73, particularly E14R, P16R, Q20R, P21R, N66R andG73R.

A particularly effective modified β-subunit of the invention comprises abasic amino acid at a position corresponding to position 4 of SEQ ID No.2, and more particularly, E4R. This substitution results in a uniqueincrease in FSH potency and expression level. The inventors have foundthat this mutation results in 2-3 fold higher production of recombinantFSH when used in combination with the other substitutions disclosedherein.

Design of FSH Superagonists

Superagonists encompassed by the present invention may be designed bycomparing the amino acid sequences of the alpha and beta FSH of interestto that of other species to identify basic residues in the proteins ofFSH of other species. Such methods are disclosed in U.S. Pat. No.6,361,992, which is herein incorporated by reference in its entirety.Consideration may also be given to the relative biological activity ofFSH from various species as to which species to chose for comparison andsubstitution. Further, homology modeling based on the structure ofrelated glycoprotein hormones is useful to identify surface-exposedamino acid residues.

Accordingly, the present invention also provides a modified FSH proteinhaving increased potency over a wild-type FSH from the same species,wherein the modified FSH comprises a basic amino acid substituted at aposition corresponding to the same amino acid position in a FSH proteinfrom another species having an increased potency over the wild-type FSHprotein. The glycoprotein being modified to increase its potency can befrom a non-human species. For example, one can compare porcine FSH tobovine FSH, design porcine FSH proteins with amino acid substitutions atpositions where the porcine and the bovine sequences are different,construct porcine FSH proteins with the selected changes, and administerthe modified porcine FSH to porcine animals. Alternatively, the FSHbeing modified can be bovine.

The present invention also provides a modified FSH having increasedpotency over the wild-type FSH from the same species, wherein themodified FSH comprises a basic amino acid substituted at a positioncorresponding to the same amino acid position in a differentglycoprotein hormone from the same species having an increased potencyover the wild-type glycoprotein hormone. For example, the beta subunitsof human FSH and human chorionic gonadotropin can be compared and aminoacid substitutions to the FSH beta subunit can be made based on anysequence divergence. Naturally, only those changes which generallyincrease the potency of the modified FSH are contemplated since thehormone receptor specificity will still need to be retained.

To modify additional amino acid positions, glycoprotein hormonesequences from human and non-humans can be aligned using standardcomputer software programs such as DNASIS (Hitachi Software EngineeringCo. Ltd.) or any of the other alignment programs listed above, includingbut not limited to ALIONment, SIM and GCG programs such as Gap, BestFit,FrameAlign and Compare. The amino acid residues that differ between thehuman and the non-human glycoprotein hormone can then be substitutedusing one of the above-mentioned techniques, and the resultantglycoprotein hormone assayed for its potency using one of theherein-mentioned assays.

The present invention also encompasses fragments of the analogsdescribed herein that have either superagonist or antagonist activity.For example, fragments of the modified alpha chains of the invention maybe used either alone or in combination with either a fragment or fulllength beta chain to create superagonist compounds. Likewise, fragmentsof the modified beta chains of the invention may be used either alone orin combination with either a fragment or full length alpha chain tocreate superagonist compounds. In some cases, fragments of the modifiedFSH molecules of the invention may also be used as antagonists, forinstance, to limit the duration of activity of an FSH therapeutic afterit has been administered.

The present invention also encompasses single chain analogs and chimericproteins incorporating the mutated regions of the analogs describedherein. For instance, the present inventors have found thatincorporation of superpotency substitutions within the alpha subunit ofdual-activity gonadotropins results in a 3-5 fold increase of bothluteotropic and follitropic activities indicating that the intrinsicactivities of dual-activity gonadotropins can be further enhanced by thecombined substitutions of the present invention. Construction ofdual-activity gonadotropins is described in U.S. Pat. No. 4,237,224,which is herein incorporated by reference in its entirety.

Characterization of FSH Superagonists

The effect of the modification or modifications to the wild-type FSHdescribed herein can be ascertained in any number of ways. For example,cyclic AMP (cAMP) production in cells transfected with a nucleic acidencoding the modified glycoprotein can be measured and compared to thecAMP production of similar cells transfected with a nucleic acidencoding the wild-type glycoprotein hormone. Alternatively, progesteroneproduction in cells transfected with the modified glycoprotein can bemeasured and compared to the progesterone production of similar cellstransfected with the wild-type glycoprotein hormone. Alternatively, theactivity of a modified glycoprotein hormone can be determined fromreceptor binding assays, from thymidine uptake assays, or from T4secretion assays. Specific examples of such assays for determining theactivity of modified glycoprotein hormones are set forth in the Examplesection contained herein. One skilled in the art can readily determineany appropriate assay to employ to determine the activity of either awild-type or a modified glycoprotein hormone.

In one embodiment of the present invention, the modified glycoproteinhormone has a potency which is increased over the potency of the wildtype glycoprotein hormone by at least about 10 fold. This increasedpotency can be assessed by any of the techniques mentioned above anddescribed in the Example contained herein, or in any other appropriateassay as readily determined by one skilled in the art. The increasedpotency does not have to be consistent from assay to assay, or from cellline to cell line, as these of course, will vary. The modified FSHmolecules of the invention may demonstrate an increase in potency of atleast about 20 fold, 30 fold, 40 fold, 50 fold, 60 fold, 70 fold, 80fold, 90 fold or even 100 fold over wild type using cell linesexpressing responsive FSH receptors at varying levels.

In another embodiment of the present invention, the modifiedglycoprotein hormone has a maximal efficacy which is increased over themaximal efficacy of the wild type glycoprotein hormone by at least about10%. This increased maximal efficacy can be assessed by any of thetechniques mentioned above and described in the Example containedherein, or in any other appropriate assay as readily determined by oneskilled in the art. The increased maximal efficacy does not have to beconsistent from assay to assay, or from cell line to cell line, as theseof course, will vary. The modified FSH molecules of the invention maydemonstrate an increase in maximal efficacy of at least about 10% fold,at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%, overwild type using cell lines expressing responsive FSH receptors atvarying levels.

Other assays suitable for characterizing the analogs described hereinare described in PCT/US99/05908, which is herein incorporated byreference in its entirety. For instance, various immunoassays may beused including but not limited to competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA, sandwichimmunoassays, immunoradiometric assays, gel diffusion precipitinreactions, immunodiffusion assays, in situ immunoassays, western blots,precipitation reactions, agglutination assays, complement fixationassays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc.

Improvements in the quality and quantity of oocytes can be assessed byin vitro and in vivo assays. It is common for improvements in oocytequantity and quality to be determined using different end points of thein vitro fertilization process such as oocyte formation, oocytefertilization, and blastocyst formation. In vitro fertilizationexperiments may follow a “superovulation protocol” in which subjects aretreated with a superactive FSH analog according to the presentinvention, which leads to the release and maturation of multipleoocytes. In in vitro fertilization experiments, FSH (superactive FSH andrecombinant wild type FSH) may be administered with hCG to triggerovulation. A control animal may be used which receives only hCG orpregnant mare serum gonadotropin (PMSG).

The quality of oocytes can be improved by increasing the fertilizationrate of oocytes in an animal. The fertilization rate of a superactivefollicle stimulating hormone can be determined in vivo or in vitro bycomparing the fertilization rate achieved with a superactive FSH to thefertilization rate achieved with the same amount of recombinant wildtype FSH. A control animal may also be used that receives hCG.

The rate of fertilization can be measured by the percent of two-cellembryos which develop per total number of oocytes. If fertilizationtakes place in vitro, two cell embryos can be counted in fertilizationdishes. In mice, two cell embryos develop approximately twenty-fourhours after fertilization.

The fertilization rate varies based on the amount of superactive FSHadministered. An animal may receive multiple does of superactive FSH.The rate of fertilization increases by at least about 10 percent as aresult of administration of superactive FSH at the maximally effectivedose for oocyte number. The rate of fertilization may increase by atleast about 20 percent, preferably at least 30 percent, 40%, 50%, 60%,70%, 80%, 90%, or 100% as a result of administration of superactive FSHat the maximally effective dose for oocyte number.

Superactive follicle stimulating hormone can improve the quality ofoocytes by improving the blastocyst formation rate per fertilizedoocyte. The rate of blastocyst formation can be measured by determiningthe percentage of two-cell embryos which form blastocysts. The rate ofblastocyst formation increases whether the blastocyst forms in vivo orin vitro. The blastocyst formation rate is dependent on the amount ofsuperactive follicle stimulating hormone administered. The rate ofblastocyst formation increases at least about 10 percent as a result ofadministration of a superactive follicle stimulating hormone at themaximally effective dose for oocyte number. The rate of blastocystformation may increase at least about 20 percent, preferably at least30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as a result of administrationof superactive FSH at the maximally effective dose for oocyte number.

Superactive follicle stimulating hormone can improve the quality ofoocytes by increasing the total number of embryos per fertilized oocyte.The increase in total number of embryos per fertilized oocyte increaseswhether fertilization occurs in vivo or in vitro. The increase in totalnumber of embryos per fertilized oocyte is dependent on the amount ofsuperactive follicle stimulating hormone administered. The total numberof embryos per fertilized oocyte increases at least about 10 percent asa result of administration of a superactive follicle stimulating hormoneat the maximally effective dose for oocyte number. The total number ofembryos per fertilized oocyte may increase by at least about 20 percent,preferably at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% as aresult of administration of superactive FSH at the maximally effectivedose for oocyte number.

Superactive FSH can be used to improve the quality and quantity ofoocytes from animals, including but not limited to, human, mouse, rat,primate, rabbit, pig, horse, sheep, and dog. Preferably, a superactiveFSH is administered to a human FSH.

FSH Analogs with Increased Serum Half-Life

The modified FSH proteins of the invention may also be further modifiedsuch that the plasma half-life is increased as compared to wild typeFSH. For instance, the modified FSH proteins of the invention mayfurther comprise at least one sequence with a potential glycosylationsite including sequences comprising N-glycosylation and/orO-glycosylation sites on either the alpha or beta chain. Sequencesproviding potential glycosylation recognition sites may be either anN-terminal or C-terminal extension on either the alpha or beta chain.Exemplary modified proteins contain an N-terminal extension on the αchain that is selected from the group consisting of ANITV (SEQ ID No. 3)and ANITVNITV (SEQ ID No. 4). Other exemplary modified proteins containa further substitution in said β chain, wherein said substitution isselected from the group consisting of Y58N and V78N.

Increased half-life may also be provided by pegylation or conjugation ofother appropriate chemical groups or by constructing fusion proteinshaving increased half life or any other method. Such methods are knownin the art, for instance as described in U.S. Pat. No. 5,612,034, U.S.Pat. No. 6,225,449, and U.S. Pat. No. 6,555,660, each of which isincorporated by reference in its entirety. Half-life may also beincreased by increasing the number of negatively charged residues withinthe molecule, for instance, the number of glutamate and/or aspartateresidues. Such alteration may be accomplished by site directedmutagenesis, with preferred alterations selected from the groupconsisting of alpha subunit substitutions A85E and A85D, among others.Such alteration may also be achieved via an insertion of an amino acidsequence containing one or more negatively charged residues into saidmodified FSH, including insertions selected from the group consisting ofGEFT (SEQ ID No. 5) and GEFTT (SEQ ID No. 6), among others. In oneembodiment, the insertion is in the alpha subunit, and is selected fromthe group consisting of APD-GEFT-VQDC (SEQ ID No. 7) and APD-GEFTT-QDC(SEQ ID No. 8), among others.

The half-life of a protein is a measurement of protein stability andindicates the time necessary for a one-half reduction in theconcentration of the protein. The serum half-life of the modified FSHmolecules described herein may be determined by any method suitable formeasuring FSH levels in samples from a subject over time, for examplebut not limited to, immunoassays using anti-FSH antibodies to measureFSH levels in serum samples taken over a period of time afteradministration of the modified FSH, or by detection of labeled FSHmolecules, i.e., radiolabeled molecules, in samples taken from a subjectafter administration of the labeled FSH.

Expression and/or Synthesis of the FSH Superagonists

The present invention also includes nucleic acids encoding the modifiedFSH α and β subunits of the invention, as well as vectors and host cellsfor expressing the nucleic acids. Appropriate promoters for theexpression of nucleic acids in different host cells are well known inthe art, and are readily interchanged depending on the vector-hostsystem used for expression. Exemplary vectors and host cells aredescribed in U.S. Pat. No. 6,361,992, which is herein incorporated byreference in its entirety.

For instance, once a nucleic acid encoding a particular glycoproteinhormone of interest, or a region of that nucleic acid, is constructed,modified, or isolated, that nucleic acid can then be cloned into anappropriate vector, which can direct the in vivo or in vitro synthesisof that wild-type and/or modified glycoprotein hormone. The vector iscontemplated to have the necessary functional elements that direct andregulate transcription of the inserted gene, or hybrid gene. Thesefunctional elements include, but are not limited to, a promoter, regionsupstream or downstream of the promoter, such as enhancers that mayregulate the transcriptional activity of the promoter, an origin ofreplication, appropriate restriction sites to facilitate cloning ofinserts adjacent to the promoter, antibiotic resistance genes or othermarkers which can serve to select for cells containing the vector or thevector containing the insert, RNA splice junctions, a transcriptiontermination region, or any other region which may serve to facilitatethe expression of the inserted gene or hybrid gene. (See generally,Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed.(1989)).

There are numerous E. coli (Escherichia coli) expression vectors knownto one of ordinary skill in the art which are useful for the expressionof the nucleic acid insert. Other microbial hosts suitable for useinclude bacilli, such as Bacillus subtilis, and otherenterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts one can also makeexpression vectors, which will typically contain expression controlsequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (Trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences for example, for initiating and completingtranscription and translation. If necessary, an amino terminalmethionine can be provided by insertion of a Met codon 5′ and in-framewith the downstream nucleic acid insert. Also, the carboxy-terminalextension of the nucleic acid insert can be removed using standardoligonucleotide mutagenesis procedures.

Additionally, yeast expression can be used. There are several advantagesto yeast expression systems. First, evidence exists that proteinsproduced in a yeast secretion systems exhibit correct disulfide pairing.Second, post-translational glycosylation is efficiently carried out byyeast secretory systems. The Saccharomyces cerevisiaepre-pro-alpha-factor leader region (encoded by the MF″-1 gene) isroutinely used to direct protein secretion from yeast. (Brake, et al.,“.varies.-Factor-Directed Synthesis and Secretion of Mature ForeignProteins in Saccharomyces cerevisiae.” Proc. Nat. Acad. Sci.,81:4642-4646 (1984)). The leader region of pre-pro-alpha-factor containsa signal peptide and a pro-segment which includes a recognition sequencefor a yeast protease encoded by the KEX2 gene: this enzyme cleaves theprecursor protein on the carboxyl side of a Lys-Arg dipeptide cleavagesignal sequence. The FSH coding sequence can be fused in-frame to thepre-pro-alpha-factor leader region. This construct is then put under thecontrol of a strong transcription promoter, such as the alcoholdehydrogenase I promoter or a glycolytic promoter. The nucleic acidcoding sequence is followed by a translation termination codon which isfollowed by transcription termination signals. Alternatively, thenucleic acid coding sequences can be fused to a second protein codingsequence, such as Sj26 or beta.-galactosidase, which may be used tofacilitate purification of the fusion protein by affinitychromatography. The insertion of protease cleavage sites to separate thecomponents of the fusion protein is applicable to constructs used forexpression in yeast. Efficient post-translational glycosolation andexpression of recombinant proteins can also be achieved in Baculovirussystems.

Mammalian cells permit the expression of proteins in an environment thatfavors important post-translational modifications such as folding andcysteine pairing, addition of complex carbohydrate structures, andsecretion of active protein. Vectors useful for the expression of activeproteins in mammalian cells are characterized by insertion of theprotein coding sequence between a strong viral promoter and apolyadenylation signal. The vectors can contain genes conferringhygromycin resistance, gentamicin resistance, or other genes orphenotypes suitable for use as selectable markers, or methotrexateresistance for gene amplification. The chimeric protein coding sequencecan be introduced into a Chinese hamster ovary (CHO) cell line using amethotrexate resistance-encoding vector, or other cell lines usingsuitable selection markers. Presence of the vector DNA in transformedcells can be confirmed by Southern blot analysis. Production of RNAcorresponding to the insert coding sequence can be confirmed by Northernblot analysis. A number of other suitable host cell lines capable ofsecreting intact human proteins have been developed in the art, andinclude the CHO cell lines, HeLa cells, myeloma cell lines, Jurkatcells, etc. Expression vectors for these cells can include expressioncontrol sequences, such as an origin of replication, a promoter, anenhancer, and necessary information processing sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Exemplary expression controlsequences are promoters derived from immunoglobulin genes, SV40,Adenovirus, Bovine Papilloma Virus, etc. The vectors containing thenucleic acid segments of interest can be transferred into the host cellby well-known methods, which vary depending on the type of cellularhost. For example, calcium chloride transformation is commonly utilizedfor prokaryotic cells, whereas calcium phosphate, DEAE dextran, orlipofectin mediated transfection or electroporation may be used forother cellular hosts.

Alternative vectors for the expression of genes in mammalian cells,those similar to those developed for the expression of humangamma-interferon, tissue plasminogen activator, clotting Factor VIII,hepatitis B virus surface antigen, protease Nexinl, and eosinophil majorbasic protein, can be employed. Further, the vector can include CMVpromoter sequences and a polyadenylation signal available for expressionof inserted nucleic acids in mammalian cells (such as COS-7).

Expression of the gene or hybrid gene can be by either in vivo or invitro. In vivo synthesis comprises transforming prokaryotic oreukaryotic cells that can serve as host cells for the vector.Alternatively, expression of the gene can occur in an in vitroexpression system. For example, in vitro transcription systems arecommercially available which are routinely used to synthesize relativelylarge amounts of mRNA. In such in vitro transcription systems, thenucleic acid encoding the glycoprotein hormone would be cloned into anexpression vector adjacent to a transcription promoter. For example, theBluescript II cloning and expression vectors contain multiple cloningsites which are flanked by strong prokaryotic transcription promoters.(Stratagene Cloning Systems, La Jolla, Cailf.). Kits are available whichcontain all the necessary reagents for in vitro synthesis of an RNA froma DNA template such as the Bluescript vectors. (Stratagene CloningSystems, La Jolla, Cailf.). RNA produced in vitro by a system such asthis can then be translated in vitro to produce the desired glycoproteinhormone. (Stratagene Cloning Systems, La Jolla, Cailf.).

Another method of producing a glycoprotein hormone is to link twopeptides or polypeptides together by protein chemistry techniques. Forexample, peptides or polypeptides can be chemically synthesized usingcurrently available laboratory equipment using either Fmoc(9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl)chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilledin the art can readily appreciate that a peptide or polypeptidecorresponding to a hybrid glycoprotein hormone can be synthesized bystandard chemical reactions. For example, a peptide or polypeptide canbe synthesized and not cleaved from its synthesis resin whereas theother fragment of a hybrid peptide can be synthesized and subsequentlycleaved from the resin, thereby exposing a terminal group which isfunctionally blocked on the other fragment. By peptide condensationreactions, these two fragments can be covalently joined via a peptidebond at their carboxyl and amino termini, respectively, to form a hybridpeptide. (Grant, G. A., “Synthetic Peptides: A User Guide,” W. H.Freeman and Co., N.Y. (1992) and Bodansky, M and Trost, B., Ed.,“Principles of Peptide Synthesis,” Springer-Verlag Inc., N.Y. (1993)).Alternatively, the peptide or polypeptide can by independentlysynthesized in vivo as described above. Once isolated, these independentpeptides or polypeptides may be linked to form a glycoprotein hormonevia similar peptide condensation reactions. For example, enzymatic orchemical ligation of cloned or synthetic peptide segments can allowrelatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen, L.,et al., Biochemistry, 30:4151 (1991); Dawson, et al., “Synthesis ofProteins by Native Chemical Ligation,” Science, 266:776-779 (1994)).

The invention also provides fragments of modified glycoprotein hormoneswhich have either superagonist or antagonist activity. The polypeptidefragments of the present invention can be recombinant proteins obtainedby cloning nucleic acids encoding the polypeptide in an expressionsystem capable of producing the polypeptide fragments thereof. Forexample, one can determine the active domain of a modified FSH proteinwhich, together with the beta subunit, can interact with a glycoproteinhormone receptor and cause a biological effect associated with theglycoprotein hormone. In one example, amino acids found to notcontribute to either the activity or the binding specificity or affinityof the glycoprotein hormone can be deleted without a loss in therespective activity.

For example, amino or carboxy-terminal amino acids can be sequentiallyremoved from either the native or the modified glycoprotein hormone andthe respective activity tested in one of many available assays describedabove. In another example, the modified proteins of the invention mayhave a portion of either amino terminal or carboxy terminal amino acids,or even an internal region of the hormone, replaced with a polypeptidefragment or other moiety, such as biotin, which can facilitate in thepurification of the modified glycoprotein hormone. For example, amodified glycoprotein can be fused to a maltose binding protein, througheither peptide chemistry of cloning the respective nucleic acidsencoding the two polypeptide fragments into an expression vector suchthat the expression of the coding region results in a hybridpolypeptide. The hybrid polypeptide can be affinity purified by passingit over an amylose affinity column, and the modified glycoprotein canthen be separated from the maltose binding region by cleaving the hybridpolypeptide with the specific protease factor Xa. (See, for example, NewEngland Biolabs Product Catalog, 1996, pg. 164)

Active fragments of the FSH molecules of the invention can also besynthesized directly or obtained by chemical or mechanical disruption oflarger glycoprotein hormone. An active fragment is defined as an aminoacid sequence of at least about 5 consecutive amino acids derived fromthe naturally occurring amino acid sequence, which has the relevantactivity, e.g., binding or regulatory activity. The fragments, whetherattached to other sequences or not, can also include insertions,deletions, substitutions, or other selected modifications of particularregions or specific amino acids residues, provided the activity of thepeptide is not significantly altered or impaired compared to themodified glycoprotein hormone. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, etc. In any case, thepeptide must possess a bioactive property, such as binding activity,regulation of binding at the binding domain, etc. Functional or activeregions of the glycoprotein hormone may be identified by mutagenesis ofa specific region of the hormone, followed by expression and testing ofthe expressed polypeptide. Such methods are readily apparent to askilled practitioner in the art and can include site-specificmutagenesis of the nucleic acid encoding the receptor. (Zoller, M. J. etal.).

The present invention also encompasses fusion proteins and chimericproteins comprising the mutations described herein, including forinstance, fusions to the CTEP domain of LH or CG proteins. Such a fusionprotein may be made by ligating the appropriate nucleic acid sequencesencoding the desired amino acid sequences to each other by methods knownin the art, in the proper coding frame, and expressing the fusionprotein by any of the means described above. Alternatively, such afusion protein may be made by protein synthesis techniques, for example,using a peptide synthesizer. The single chain analogs and chimericproteins of the invention may incorporate a peptide linker between thealpha and beta subunits, or between different portions of the chimericprotein.

Methods of Treatment

The modified FSH superagonists of the present invention may be used totreat any condition associated with glycoprotein hormone activity.Conditions “associated with glycoprotein hormone activity” are ones thatare either completely or partially caused by altered glycoproteinhormone responsiveness, or ones that benefit from the administration ofglycoprotein hormone. For instance, such conditions include, but are notlimited to ovulatory dysfunction, luteal phase defects, unexplainedinfertility, male factor infertility, time-limited conception, low FSHreceptor expression, low FSH receptor sensitivity, FSH receptor bindingdeficiencies, FSH receptor coupling deficiencies, low testosteroneproduction, male pattern baldness, and pituitary failure or injury.

In particular, the quantity and quality of oocytes can be improved byadministering a superactive FSH analog as described herein to an animal.For example, as reported herein, Applicants have surprisingly found thatby administering a superactive FSH containing a modified alpha-subunitwith basic amino acids at position 13, 14, 16 and 20, a dramaticincrease in the quantity and quality of oocytes is obtained. The effectsof a superactive FSH on oocyte quantity and quality may be furtherenhanced by increasing the FSH serum half-life of the superactive FSH.The FSH serum half-life can be increased by further modifying thesuperactive FSH. Further modifications, including but not limited tothose previously described, can be used to increase FSH serum half-life.For instance, an ANITV (SEQ ID No. 3) extension may be used to prolongFSH serum half-life.

According to U.S. Pat. No. 5,574,011, herein incorporated by referencein its entirety, FSH stimulates the gonads to produce steroids, such astestosterone. Accordingly, the FSH analogs of the invention could beused to treat any condition associated with low steroid production, andparticularly low testosterone production. According to U.S. Pat. No.6,562,790, herein incorporated by reference, coronary artery blockage istreatable with testosterone. Therefore, the analogs of the presentinvention may be used to elevate testosterone levels in patientsexhibiting coronary artery disease.

The analogs of the present invention may also be used in therapeuticregimens of assisted reproduction in either a male or female subjectcomprising administering an assisting amount of the modified FSH to thesubject. In such methods, the analogs may be administered alone or incombination with other therapeutics, for instance, including but notlimited to Clomiphene citrate, GnRH (gonotropin releasing hormone) andLH (Luteinizing hormone). For example, in a subject with isolatedgonadotropin deficiency (IGD), administration of modified FSH and LH maybe administered to the subject to restore normal gonadal function. It iswidely known in the art that glycoprotein hormones such as FSH and LHare integral in female reproductive physiology, and these glycoproteinhormones may be administered to a subject to overcome a number ofreproductive disorders and thereby assist reproduction.

The analogs of the invention are particularly useful for treating womenprone to ovarian hyperstimulation, for instance by using analogs havingdifferent serum half-lives in a combined regimen. Such methods mayinclude (a) administering an assisting amount of a first modified FSHaccording to the invention wherein the plasma half-life of said firstmodified FSH is increased as compared to wild type FSH, and (b)subsequently administering an assisting amount of a second modified FSHaccording to the invention wherein the plasma half-life of said secondmodified FSH is decreased as compared to said first modified FSH. Forinstance, analogs demonstrating decreased half-life as compared towild-type FSH, i.e. TR-4401, may be useful for treating women prone toovarian hyperstimulation.

A skilled practitioner in the art can readily determine the effectiveamount of the glycoprotein hormone to administer and will depend onfactors such as weight, size, the severity of the specific condition,and the type of subject itself. The therapeutically effective amount canreadily be determined by routine optimization procedures. The presentinvention provides glycoprotein hormones with increased potency relativeto the wild-type glycoprotein hormone. These modified glycoproteinhormones will allow a skilled practitioner to administer a lower dose ofa modified glycoprotein hormone relative to the wild-type glycoproteinhormones to achieve a similar therapeutic effect, or alternatively,administer a dose of the modified glycoprotein hormone similar to thedose of the wild-type glycoprotein hormone to achieve an increasedtherapeutic effect.

Depending on whether the glycoprotein hormone is administered orally,parenterally, or otherwise, the administration of the prostaglandin canbe in the form of solid, semi-solid, or liquid dosage forms, such as,for example, tablets, pills, capsules, powders, liquids, creams, andsuspensions, or the like, preferably in unit dosage form suitable fordelivery of a precise dosage. The glycoprotein hormone may include aneffective amount of the selected glycoprotein hormone in combinationwith a pharmaceutically acceptable carrier and, in addition, may includeother medicinal agents, pharmaceutical agents, carriers, adjuvants,diluents, etc. By “pharmaceutically acceptable” is meant a material thatis not biologically or otherwise undesirable, i.e., the material may beadministered to an individual along with the selected glycoproteinhormone without causing unacceptable biological effects or interactingin an unacceptable manner with the glycoprotein hormone. Actual methodsof preparing such dosage forms are known, or will be apparent, to thoseskilled in this art; for example, see Remington's PharmaceuticalSciences, latest edition (Mack Publishing Co., Easton, Pa.).

Genetic therapy is another approach for treating hormone disorders withthe modified glycoprotein hormones of the present invention. In thisapproach, a gene encoding the modified glycoprotein hormone can beintroduced into a cell, such as a germ line cell or a somatic cell, sothat the gene is expressed in the cell and subsequent generations ofthose cells are capable of expressing the introduced gene. For example,a nucleic acid encoding a modified FSH protein of the invention can beinserted into an ovarian cell, or its precursor, to enhance ovulation.Suitable vectors to deliver the coding sequence are well known in theart. For example, the vector could be viral, such as adenoviral,adenoassociated virus, retrovirus, or non-viral, such as cationicliposomes.

The analogs of the present invention have an enhanced activity over wildtype protein and are therefore particularly suitable for deliveringagents to cells expressing glycoprotein hormone receptors. Accordingly,the present invention further provides a method of delivering an agentto a cell expressing a glycoprotein receptor in a subject in needthereof using the modified glycoprotein hormones of the invention. Themethod of delivering an agent to a cell (i.e. targeted delivery) canemploy any suitable agent, depending on the nature of the subject'sillness or suspected illness. The agent can be a cytoprotectivecompound, antibody, drug, sensitizer, biological response modifier,radionuclide, toxin or combination thereof.

In certain embodiments, the methods of targeted delivery are for thetreatment of a subject with a disorder or suspected disorder associatedwith abnormal glycoprotein receptor expression. In certain embodiments,the methods of targeted delivery are for the diagnosis or detection of adisorder associated with abnormal glycoprotein receptor expression. Incertain embodiments, the methods of targeted delivery can be used inconjunction with other therapies, diagnostic procedures or clinicalmodalities, including radiation and/or surgery.

In one embodiment, the methods provide for targeted delivery of anagent, wherein the agent is a cytoprotective compound. Cytoprotectivecompounds are those compounds which act to protect or decrease theincidence or severity of injury to a cell. Commercially availablecytoprotective compounds include mesna (MESNEX®, Bristol-Myers Squibb),amifostine (ETHYOL®, Alza), dexrazoxane (ZINECARD®, Pharmacia & Upjohn)and leucovorin (multiple manufacturers).

In one embodiment, the agent can be any drug used to treat various formsof cancer, such as, for example, natural or synthetic estrogens,estrogen receptor modulators, progestins, androgens,gonadotropin-releasing hormones, androgen inhibitors, bisphosphonates,glucocorticoids, thyroid hormones, antithyroid agents, iodine agents,bromocriptine, alkylating agents, antimetabolites, antimitotic agents,epipodophyllotoxins, antineoplastic antibiotics, antineoplastichormones, platinum coordination complex agents, anthracenediones,substituted ureas, methylhydrazine derivatives, DNA topoisomeraseinhibitors, retinoids, porfimer, mitotane or combinations thereof.

In one embodiment, the agent can be any drug used to treat cancers ofthe male or female reproductive systems (e.g. endometrial cancer,uterine cancer, cervical cancer, breast cancer, testicular cancer). In apreferred embodiment, the agent can be clomiphene, finasteride,propylthiouracil, methimazole, bleomycin, vincristine, vinblastine,cisplatin, mitomycin, ifosfamide, cyclophosphamide, doxorubicin,paclitaxel, fluorouracil, carboplatin, epirubicin, altretamine,vinorelbine, mitoxantrone, prednisone or combinations thereof.

Drugs known to enhance the cytotoxic effect of certain anti-cancer drugsand radiopharmaceuticals can also be used. Such drugs are commonlyreferred to as sensitizers. Examples of sensitizers which enhance theactivity of various therapeutic drugs (e.g., anti-cancer drugs) arebuthionine sulfoximine and calcium channel blockers such as verapamil,and diltiazem. (See, U.S. Pat. No. 4,628,047 and Important Advances inOncology 1986, DeVita, et al., Eds., J. B. Lippincott Co., Philadelphia,pages 146-157 (1986), incorporated herein by reference in theirentireties.) Other sensitizers known in the art are metronidazole,misonidazole, certain 2-sulfamyl-6-nitrobenzoic acid derivatives,2,6-disubstituted derivatives of 3-nitropyrazine, and certainisoindoledione compounds. (See, U.S. Pat. Nos. 4,647,588; 4,654,369;4,609,659 and 4,494,547, incorporated herein by reference in theirentireties.)

In certain embodiments, the agent can be a biological response modifier.Any biological response modifier can be used in the scope of theinvention. Examples of biological response modifiers useful in themethods of the invention include, but are not limited to interferon-α,interferon-β, interferon-γ, tumor necrosis factor, lymphotoxin,interleukin-1, interleukin-2, interleukin-3, interleukin-4,interleukin-5, interleukin-6 or combinations thereof.

In certain embodiments, the agent can be an antibody. The antibody canbe a monoclonal or polyclonal antibody. In certain embodiments, theantibodies can be humanized antibodies, chimeric antibodies, orfunctional antibody fragments including, for example, Fab1, Fab2, etc.

Examples of toxins which can be employed in the methods of the inventionare ricin, abrin, diphtheria toxin, Pseudomonas exotoxin A, ribosomalinactivating proteins, and mycotoxins; e.g., trichothecenes.Trichothecenes are a species of mycotoxins produced by soil fungi of theclass fungi imperfecti or isolated from Baccharus megapotamica (Bamburg,Proc. Molec. Subcell Bio. 1983, 8:41-110, Jarvis and Mazzola, Acc. Chem.Res. 1982, 15:338-395, incorporated herein by reference in theirentireties.) Therapeutically effective modified toxins or fragmentsthereof, such as those produced through genetic engineering or proteinengineering techniques, can be used.

Any means of coupling or linking an agent to a modified glycoproteinhormone can be employed. For example a number of different cleavablelinkers have been described previously. See, U.S. Pat. Nos. 4,618,492;4,542,225; and 4,625,014, incorporated herein by reference in theirentireties. The mechanisms for release of an agent from these linkergroups include by irradiation of a photolabile bond, and acid-catalyzedhydrolysis. U.S. Pat. No. 5,563,250, incorporated herein by reference inits entirety, discloses immunoconjugates comprising linkers of specifiedchemical structure, wherein the linkage is cleaved in vivo, releasingthe compound (radiopharmaceutical, drug, toxin, etc.) in its nativeform. The linker is susceptible to cleavage at mildly acidic pH, and isbelieved to be cleaved during transport into the cytoplasm of a targetcell, thereby releasing the biologically active compound inside a targetcell. U.S. Pat. No. 4,671,958, incorporated herein by reference in itsentirety, includes a description of immunoconjugates comprising linkerswhich are cleaved at the target site in vivo by the proteolytic enzymesof the patient's complement system.

Other means of coupling or linking have been described. For example,linker molecules are commercially available, such as those availablefrom Pierce Chemical Company, Rockford, Ill. (See the Pierce 1986-87General Catalog, pages 313-354, incorporated herein by reference in itsentirety.) Means for coupling to an antibody, (See, for example, U.S.Pat. No. 4,671,958 and U.S. Pat. No. 4,659,839, incorporated herein byreference in their entireties) and means of linking or couplingradionuclide metal chelates, toxins and drugs to proteins are known.See, for example, European Patent Application Publication No. 188,256;U.S. Pat. Nos. 4,671,958; 4,659,839, 4,414,148; 4,699,784; 4,680,338;4,569,789; and U.S. Pat. No. 4,590,071; Borlinghaus et al. Canc. Res.47:4071-4075, Aug. 1, 1987, Foran, Best Pract. Res. Clin. Haematol.2002, 15(3): 449-65 and Fotiou, et al., Eur. J. Gynaecol. Oncol. 1988,9(4): 304-7 incorporated herein by reference in their entireties. Inview of the large number of methods that have been reported for couplinga variety of radiodiagnostic compounds, radiopharmaceuticals, drugs,toxins, and other agents to proteins, one skilled in the art will beable to determine a suitable method for attaching a given agent to amodified glycoprotein.

Methods of Imaging

The analogs of the present invention have an enhanced activity over wildtype protein and are therefore particularly suitable for imaging cellsexpressing glycoprotein hormone receptors. Accordingly, in oneembodiment, the invention further provides methods of imaging cellscomprising a glycoprotein hormone receptor using the modifiedglycoprotein hormones of the present invention. The method of imagingand detecting the hormone can be any method known to those of skill inthe art. Commonly used imaging methods include, for example, magneticresonance imaging (MRI), X-ray, computed tomography (CT), positronemission tomography (PET), mammography and ultrasound.

Methods of imaging subjects using basic radiologic techniques have beendescribed, for example, “Textbook of Radiology and Imaging,” Sutton andLivingstone, 7th Edition, (2 Volume set), Churchill Livingstone(Elsevier Sciences), London, 2002, “A Concise Textbook of Radiology,”Armstrong and Wastie (eds.) Arnold Publishing (The Thomson Corporation),Scarborough, Ontario, Canada, 2001, “Walter & Miller's Textbook ofRadiotherapy,” Bomford and Knuckler, 6th Edition, Churchill Livingstone(Elsevier Sciences), London, 2001, incorporated herein by reference intheir entireties. See also, Bottomley, Comput. Radiol. 1984, 8(2):57-77, Dixon, Radiology 1984, 153(1):189-94, Daley and Cohen, CancerRes. 1989, 49(4):770-9, Ellis, et al., Clin. Radiol. 2001, 56(9):691-9,Paushter, et al., Med. Clin. North Am. 1984, 68(6):1393-421, Blecher,Aust. Fam. Physician 1983 12(6):449-50, 452, Bragg, Cancer 1977, 40(1Suppl):500-8, Moseley, Br. Med. J. (Clin. Res. Ed.) 1982,284(6323):1141-4, Lentle and Aldrich, Lancet 1997, 350(9073):280-5,Weber, et al., Strahlenther Onkol. 1999, 75(8):356-73, Hanbidge, Can. J.Gastroenterol. 2002, 16(2):101-5, Miles, Eur. Radiol. 2003, Suppl5:M134-8, Prigent-Le Jeune, et al., Eur. J. Nucl. Med. Mol. Imaging2004, Feb. 19 [Epub ahead of print], DeSimone, et al., Gynecol. Oncol.2003, 89(3):543-8 and Goldenberg, et al., J. Clin. Oncol. 1987,5(11):1827-35, incorporated herein by reference in their entireties.

Any suitable means of imaging or detecting can be employed, depending,inter alia, on the nature of the subject's disorder or suspecteddisorder, the tissue to be imaged and whether functional (physiologic)or structural (anatomic) images are desired. In some embodiments, amongothers, the methods of imaging provide that detecting an amount of alabeled modified glycoprotein hormone in a subject or detectingincreased levels of a modified glycoprotein hormone in a subjectindicates the presence of an autoimmune disorder or a cancerous disorderselected from the group consisting of ovarian cancer, uterine cancer,cervical cancer, endometrial cancer, breast cancer, testicular cancer orpituitary tumor.

Imaging methods can be broadly categorized as those that provideinformation regarding the structure or anatomy of a subject or thosethat provide function or physiology of a subject. Structural imagingprovides the shape of a bone or tissue component to determine if thereare abnormal formations or destruction of certain elements. Tumors orthe presence of cancerous cells can appear as structural changes. Anewer type of structural imaging provides the chemical composition ofdifferent parts of a tissue in order to determine if there is ongoinginjury or abnormal biochemical processes (e.g. presence or growth ofcancerous cells). See, for example, Bonilha, et al., Med. Sci. Monit.2004, 10(3):RA40-6, epub 2004 Mar. 1, Ballmaier, et al., Psychiatry Res.2004, 15; 130(1):43-55, Ballmaier, et al., Biol. Psychiatry, 2004:55(4):382-9, Cha, Magn. Reson. Imaging Clin. N. Am. 2003, 11(3):403-13and Kopelman, et al., Hippocampus, 2003; 13(8):879-91, incorporatedherein by reference in their entireties.

Functional imaging is a relatively new technique which seeks toascertain whether particular tissues or organs are performing particularfunctional tasks. This technique can capitalize on a number ofphysiologic processes, including, for example, blood flow andactivity-associated with changes in blood flow (i.e. neoplastic presenceor growth) and monitoring responses to chemotherapy. See, for example,Takeuchi, et al., J. Med. Invest. 2004, 51(1-2):59-62, Otsuka, et al.,J. Med. Invest. 2004, 51(1-2):14-9, Martincich, et al., Breast CancerRes. Treat. 2004, 83(1):67-76, Cohen and Goadsby, Curr. Neurol.Neurosci. Rep. 2004, 4(2):105-10 and Lewis, et al., Eur. J. Neurosci.2004, 19(3):755-60, incorporated herein by reference in theirentireties.

In general, radiological methods such as, for example, magneticresonance imaging (MRI), X-ray, computed tomography (CT), mammographyand ultrasound provide structural or anatomic information regarding asubject. Radiological methods such as, for example, nuclear medicine,radionuclide imaging and positron emission tomography (PET) providefunctional or physiologic information regarding a subject. Bothstructural and functional imaging are within the scope of the presentinvention.

In one embodiment of the invention, the imaging methods provide that themodified glycoprotein hormone is labeled (i.e. a contrast agent isused). Any label or contrast agent can be used. See, Minato, et al. J.Comput. Assist. Tomogr. 2004, 28(1):46-51, Antoch, et al., JAMA 2003,290(24):3199-206, Brinker, Rev. Cardiovasc. Med. 2003; 4 Suppl 5:S19-27,el-Diasty, et al., J. Urol. 2004, 171(1):31-4, Williams, et al., Int. J.Oral Maxillofac. Surg. 2003, 32(6):651-2, Follen, et al., Cancer 2003,98(9 Suppl):2028-38, Behrenbruch, et al., Med. Image Anal. 2003,7(3):311-40, Knopp, et al., Mol. Cancer Ther. 2003, 2(4):419-26,incorporated herein by reference in their entireties. The label can beany label known to those of skill in the art. In one embodiment, thelabel can be a radiopaque label, radioactive label, fluorescence labelor paramagnetic label.

Radionuclides generally emit either beta (β) or gamma (γ) radiation.I131 emits about 90% β-radiation and about 10% γ-particles has aphysical half life of about 8 days. Tc99m emits γ-radiation and has ahalf life of about 6 hours. Following administration of, for example, aTc99m-labeled protein, the biodistribution of the radionuclide can bedetected by scanning the patient with a gamma camera using knownprocedures. Accumulations of Tc99m at the target site(s) is thus easilyimaged. See, Toohey, Radiographics. 2000; 20:533-546, Kostakoglu, etal., RadioGraphics 2003, 23:315-340, Saremi, et al., RadioGraphics 2002,22:477-490, Intenzo, et al., RadioGraphics 2001, 21:957-964, Ranger,RadioGraphics 1999, 19:481-502, Simpkin, RadioGraphics 1999, 19:155-167,Janoki and Kerekes, Acta Physiol. Hung. 1992, 79(2):183-96, Hoefnagel,Anticancer Drugs 1991, 2(2):107-32, Hoefnagel, Eur. J. Nucl. Med. 1991,18(6):408-31, Gatley, et al., Acta Radiol. Suppl. 1990, 374:7-11, Ott,Br. J. Radiol. 1989, 62(737):421-32, Andersen, Cerebrovasc. Brain Metab.Rev. 1989, 1(4):288-318 and Miraldi, Int. J. Radiat. Oncol. Biol. Phys.1986, 12(7):1033-9, incorporated herein by reference in theirentireties.

In addition to I131 or Tc99m, any radioisotope known to those of skillin the art can be employed in the methods of the invention. Otherradionuclides and chelates can include, for example, Co57, Co58, Cr51,F18 FDG, Ga67, In111 chloride, In111 pentetate (DTPA), In111oxyquinoline(oxine), In111 Capromab pendetide, In111 Imciroma pentetate, In111,pentetreotide, In111 satumomab pendetide, I 123, I125 iothalamate, I125human serum albumin (RISA), I131 iodohippurate, I131iodomethylnorcholesterol (NP-59), I131 metaiodobenzylguanidine (MIBG),Kr81m gas, P32 chromic phosphate, P32 sodium phosphate, Ru82, Sm 153lexidronam (Sm-153 EDTMP), Sr89, Tl 201 and Xe133.

Diagnostic Assays

The present invention further provides for the detection of analytesthat interfere with the binding of the modified glycoprotein hormones ofthe invention to a glycoprotein hormone receptor. In one embodiment, themethods provide for the detection of an analyte that interferes with thebinding of a modified glycoprotein hormone receptor in a biologicalsample, said method comprising (I) contacting the sample with a modifiedglycoprotein hormone according to the present invention and (ii)detecting a signal wherein the presence or amount of the signal detectedindicates the presence or absence of an analyte that interferes with thebinding of a modified glycoprotein hormone to a glycoprotein receptor.

In one embodiment, the method for the detection of an analyte is acompetitive binding assay. A competitive binding assay is an assay basedon the competition between a labeled and an unlabelled ligand in thereaction with a receptor binding agent (e.g. antibody, receptor,transport protein). IUPAC Compendium of Chemical Terminology, 1997, 2ndedition, “Competitive Protein Binding Assays” Odell and Daughaday, W. H.Lippincott, 1972 and “Principles of Competitive Protein-binding Assays”Odell and Franchimont, P. John Wiley & Sons Inc., 1983, incorporatedherein by reference in their entireties. See also, U.S. Pat. No.6,537,1760, incorporated herein by reference in its entirety.

In certain embodiments, the signal is the presence or amount of themodified glycoprotein hormone bound with the glycoprotein receptor inthe sample. In certain embodiments, the method employs the detection ofa secondary signal, such as, for example, the detection of the presenceor amount of cAMP or a steroid (e.g. progesterone). In certainembodiments, the methods employ the use of whole cells in the biologicalsample. In certain embodiments, the methods employ only parts of cells,for example, cell membranes.

In certain embodiments, the assay can be performed in solution. Incertain embodiments, one or more components of the assay can beimmobilized on a solid phase. Plastic surfaces, microparticles, magneticparticles, filters, polymer gel materials and other solid-phasesubstrates can be used as solid phases. See, for example, U.S. Pat. Nos.6,664,114; 6,589,798; 6,479,296 and 6,294,342, incorporated herein byreference in their entireties. It is possible to automate the methods ofassay provided in the invention.

Methods of Designing Glycoprotein Receptor Agonists and AntagonistsUsing FSH Superagonists

The present invention also provides methods of designing new receptoragonists and antagonists based on the interaction of the FSH proteins ofthe invention and a cognate receptor. Such methods involve predictinginteractions of charge motifs in the FSH proteins of the invention withcomplementary amino acid residues within a cognate receptor. Forinstance, such a method may involve comparing the differences ininteraction in terms of binding and bioactivity of FSH to receptors fromevolutionary distant species, e.g., human LH versus rat LH receptor,localizing charged amino acids within extracellular domains and/orextracellular loops that are present in only one of the two receptorsequences, performing alanine scanning and charge reversal mutagenesisto further validate given prediction, building a model ofhormone-receptor complex incorporating validated interactions, anddesigning new hormone analogs and receptor antagonists using the model.New hormone analogs include those that are predicted to bind to thereceptor using the model. New antagonists include those that aredesigned from the domains and/or loop of the receptor protein that arepredicted to bind to the FSH analog using the model.

For example, one of the analogs of the invention (TR-4402, comprisingthe substitutions alpha(E14R+Q20R+G73R)+betaE4R), has been found tointeract with the rat LH receptor (SEQ ID No. 23, NCBI Accession No.NP_037110) at high concentrations, but not the human LH receptor (SEQ IDNo. 24, NCBI Accession No. NP_000224, data not shown). Based on thedifference in specificity of TR-4402 at these receptors, Arg14, Arg20and Arg73 should interact with negatively charged residues Asp and Gluin the rat LH receptor. The negatively charged residues present in therat receptor but absent in the human receptor are Asp 312 and Glu 314(based on the human LH receptor amino acid sequence with signal peptide)(Ser and Lys, respectively, in the human LH receptor). The correspondingresidues in the human FSH receptor (SEQ ID No. 22, NCBI Accession No.AAA52477) are Glu316, Asp317 and Glu319. This cluster of acidic aminoacids, therefore, is predicted to interact with Arg14, Arg20 and Arg73of the alpha subunit of TR-4402. This information should allow forbetter modeling of glycoprotein hormone interactions, and willcontribute to the design of new glycoprotein analogs, includingpeptide/protein antagonists containing the sequence corresponding to298-338 of the human FSH receptor and including Glu300 and Asp302.

The following examples are provided to describe and illustrate thepresent invention. As such, they should not be construed to limit thescope of the invention. Those in the art will well appreciate that manyother embodiments also fall within the scope of the invention, as it isdescribed hereinabove and in the claims.

Examples Example 1 Production and Characterization of FSH Superagonists

Site Directed Mutagenesis. Site directed mutagenesis of human alpha (SEQID No. 1) and FSH beta (SEQ ID No. 2) subunit cDNA was performed usingQuickChange Mutagenesis Kit from Stratagene. Analogs were designedaccording to the methods described in U.S. Pat. No. 6,361,992, hereinincorporated by reference in its entirety.

After subcloning into the expression vectors, the entire PCR products ofall constructs were sequenced to verify the mutations and to rule outany undesired polymerase errors.

Transient Expression. Analogs were expressed transiently in Chinesehamster ovary (CHO-K1) cells. Cells were transiently co-transfected in60 or 100 min culture dishes with wild-type or mutant subunit cDNAs(alpha and FSH beta), using a transient transfection protocol based on aliposome formulation (LipofectAMiNE reagent, Gibco BRL). After recoveryfor 12 hours in regular growth medium, transfected cells were culturedin CHO-serum free medium (CHO-SFM, Gibco BRL) for 72 hours.Subsequently, the conditioned media including control medium from mocktransfections using the expression plasmids without gene inserts, wereharvested, concentrated with Centriprep 10 concentrators (Amicon,Beverly, Mass.) and stored at −70° C. Analogs were quantitated with apanel of different monoclonal and polyclonal antibodies recognizingdifferent FSH epitopes.

FSH Bioactivity Assay. The follitropic activity of the analogs wasassessed by their ability to induce cAMP production in CHO cellsexpressing hFSH receptors. CHO cells stably expressing the hFSH receptorwere grown to confluence in 96-well tissue culture plates. Subsequently,cells were incubated either in salt-free conditions (2 h) or withphysiological media (1 h) at 37° C., 5% CO2 with serial dilutions ofwild-type and mutant FSH as well as control medium from mocktransfections. The amount of cAMP produced was determined byradioimmunoassay.

FSH mutations showing the highest bioactivity in vitro and no adverseeffects on FSH production were chosen for combination strains. FIG. 1includes graphs showing a comparison of the effect of various singlemutations on FSH bioactivity in vitro, as measured using transienttransfection of CHO-FSHR cells. Single mutations showing the highestpotency included basic substitutions at alpha positions Q13, E14, V68,P21 and G73, and at beta position E4. An arginine substitution at F18resulted in a loss of bioactivity. Beta E4R in particular resulted inenhanced FSH production (see FIG. 2). The synergistic effect onbioactivity of several combined substitutions is shown in FIG. 3.

In total, 26 single mutations in the alpha subunit and 23 singlemutations in the beta subunit were tested, and the top mutations in eachsubunit were selected to construct lead analogs with combinedsubstitutions. Table 1 below shows combined mutations with a provenincrease in bioactivity in vitro.

TABLE 1 Combined Substitutions Resulting in Enhanced Potency AnalogSubstitutions 4201 alphaE14R + betaE4R 4202 alpha(E14R + N66R) + WT beta4203 alpha(E14R + G73R) + WT beta 4204 alpha(P16R + Q20R) + WT beta 4205alpha(Q20R + P21R) + WT beta 4301 alpha(E14R + Q20R + G73R) + WT beta4302 alpha(E14R + P21R + G73R) + WT beta 4303 alpha(E14R + N66R +G73R) + WT beta 4304 alpha(E14R + N66R) + betaE4R 4305 alpha(E14R +G73R) + betaE4R 4306 alpha(P16R + Q20D + P21R) + WT beta 4307alpha(P16R + Q20R + P21R) + WT beta 4308 alpha(N66K + G73K + A81K) + WTbeta 4401 alpha(Q13R + E14R + P16R + Q20R) + WT beta 4402 alpha(E14R +Q20R + G73R) + betaE4R 4403 alpha(E14R + P21R + G73R) + betaE4R 4404alpha(E14R + N66R + G73R) + betaE4R 4405 alpha(Q13K + E14K + P16K +Q20K) + WT beta 4501 alpha(E14R + Q20R + P21R + N66R + G73R) + WT beta4601 alpha(Q13K + E14K + P16K + Q20K + N66K + G73K) + WT beta 4602alpha(E14R + P16R + Q20R + P21R + N66R + G73R) + WT beta 4603alpha(E14R + Q20R + P21R + N66R + G73R) + betaE4R 4701 alpha(E14R +P16R + Q20R + P21R + N66R + G73R) + betaE4R 4901 [alpha(E14R + Q20R +G73R) + betaE4R] with N-terminal ANITV (SEQ ID No. 3) extension in thealpha subunit 4910 [alpha(Q13R + E14R + P16R + Q20R) + WT beta] withN-terminal ANITV (SEQ ID No. 3)extension in the alpha subunit

As known in the art, a particular drug may exhibit differentefficiencies depending on the system used. See Kenakin, “PredictingTherapeutic Value in the Lead Optimization Phase of Drug Disclovery,”Nature Rev. 2: 429-38 (2003). Therefore, the bioactivity of the analogsof the invention was also tested using rat granulosa cells expressinglow quantities of FSH receptor (GLHR-15 cells). As shown in the graphsin FIG. 4, the analogs of the invention still resulted in significantcAMP responses, as compared to wild type FSH and free alpha chain, whichdid not show a significant dose dependent response.

Example 2 Purification and Characterization of Analog TR-4402

Analog TR-4402 was chosen for purification and further characterization.TR-4402 is a FSH analog containing two mutations in the alpha L1 loop(αE14R and αQ20R), one mutation in the alpha L3 loop (αG73R), and onemutation in the beta L1 loop (βE4R). See FIG. 5. The cell line producingTR-4402 was established by co-transfection of a modified pED vectorcontaining the cDNA of the human alpha subunit and dihydrofolatereductase (DHFR) gene, and a modified pIRES vector containing the cDNAof FSH beta subunit, separated by an IRES sequence and the amplifiablegene marker, adenosine deaminase (ADA), into CHO-DHFR(−) bylipofectamine method. Transfected cells were cultured in selectionmedium (ribonucleosides and deoxyribonucleosides deficient aMEM with 10%dialyzed FBS). For stable production of hFSH analogs in a CHO doubledeletion mutant (dhfr−/dhfr−), the CHO-DG44 cell line was kindlyprovided by Dr. L. Chasin (Columbia University, New York, N.Y.).

Clonal cell lines secreting FSH#4402 were cultured in increasingconcentrations of methotrexate (MTX) up to 2 μM. DHFR amplification isbased on systematic increases of MTX in medium without addednucleosides. Cells were qualified for next amplification step afterregaining their polygonal morphology (2-3 weeks). Since theconcentration of MTX increased about 800× (from 0.005 uM to about 4 μM)the amplification process took about 4 months. Clones with highestsecretion level were also subjected to a second treatment, involving theutilization of deoxycoformycin, directed to amplify the ADA marker gene.

Single clones were tested for expression using FSH immunoassayestablished for detection of FSH#4402. A stable cell line (clone ID:H-2-3), transfected with pED-analog a+pIRES-ADA-analog b (molar ratio ofa:b=1:5, 5 mg total DNA), was selected and propagated in alpha-minimumessential medium (a-MEM: Cat #: 12561-056, Lot #1141509, withL-glutamine, without ribonucleosides and deoxyribonucleosides); GIBCO,Grand Island, N.Y.), supplemented with 10% dialyzed fetal bovine serum(Gibco, Cat No: 26400-044) and 2 mM methotrexate (MTX; ICN, Cat. No:102299, Aurora, Ohio).

Preparation of bioreactor inoculums. When >90% CHO-DG44 cells wereconfluent in the 500-cm2 T-flasks, the cells were trypsinized,centrifuged at 400 g for 5 min at 4° C. Total cells (1.6 ′109/500 ml ofa-MEM culture medium) were inoculated into the Celligen plus Bioreactorby feeding cell suspension in a 2 L-plastic bottle.

After amplification, the cell line producing the highest amount ofFSH#4402 was grown in multiple flasks. Cells were further propagatedusing perfusion mode in Packed-Bed Bioreactor 3.5 L Bioreactor withinternal retention device (basket) and vertical mixing system (CelligenPlus, New Brunswick Scientific, Edison, N.J.). Cells were trapped ontoFibracel disks located inside the retention assembly. Dissolved oxygenwas kept at 50% saturation. Temperature was 37 C. Agitation was 100 rpm.The pH 7.2 was maintained using a four-gas mixing system and automaticinjection of sodium bicarbonate. Perfusion was adjusted to keep glucoselevel above 1.5 g/L and lactate below 1.5 g/L.

FBS weaning process and FSH-TR 4402 analog production in Celligen plusbioreactor. CHO III A (Gibco, Formula No.: 97-0147DK, Lot No.: 1147268)supplemented with hypoxanthine-thymidine supplement (Gibco, 100′),penicillin-streptomycin (Gibco, 100′; Cat #: 15140-122, Lot #: 1161387),glutamax-1 (Gibco, 100′; Cat #: 35050-061, Lot #: 1163550), 10% pluronicF-68 (Gibco, 100′, Cat #: 24040-032, Lot #: 1153058), and 1% dialyzedfetal bovine serum (Gibco) was used for serum weaning in the bioreactor.At day 17 of the bioreactor operation, we changed CHO-III A culturemedium to CHO protein free, animal component-free medium (Sigma, Cat #:C-8730 Lot No.: 122K8401) until day 23 (see SLIDE summarizing bioreactorrun). Medium from bioreactor was harvested, centrifuged, filtered (0.45μm membrane) and concentrated using Millipore concentrators.

Purification. TR-4402 was purified using immunoaffinity (monoclonal AbME.112 from Maine Biotechnology Services, Inc.) and hydrophobicinteraction chromatography. Purity was assessed based on SDS-PAGE(˜85%).

Modified analog TR-4402 was characterized using in vitro bioassaysemploying CHO, human granulosa-like tumor (KGN) and rat granulosa(GLHR-15) cell lines expressing human FSH receptor and total cAMPproduction as an end point (see FIGS. 6-8). Using CHO-FSHR cells,TR-4402 showed a 30 fold increase in potency and 17% increase in Vmax ascompared to Follistim (wild type FSH). Using KGN-FSHR, TR-4402 alsoshowed a 30 fold increase in potency as compared to wild type FSH.

TR-4402 was also tested for binding to LH and TSH receptors to confirmFSH analog specificity, and isoelectric focusing was employed to confirmcarbohydrate chain heterogeneity, i.e., the presence of alpha and betasubunits (data not shown).

The effect of TR-4402 as compared to wild type FSH was also tested onmouse follicles in vitro using EggCentris in vitro follicle bioassay.This assay studies the growth and development of early preantralfollicles up to the ovulatory stage. The whole in vitro, process closelymimics the physiology of in vivo folliculogenesis. The culture systembegins with isolation of a homogenous class of mouse preantral folliclesbetween 100 and 130 μM in diameter. The follicules were individuallyplated and cultured for 12 days with 1, 3 and 9 mIU/mL of wild type(“compound 3”) and TR-4402 (“compound 4”).

The follicle bioassay indicated that quality of the oocytes is improvedafter exposure to TR-4402 in comparison to Follistim (wild type) asshown by enhanced follicle survival (FIG. 9), enhanced antrum formation(FIG. 10), enhanced mucification of COC (FIG. 11), enhanced nuclearmaturation (FIG. 12) and enhanced progesterone production (FIG. 13).Such differences could be related to the presence and anti-apoptoticaction of FSH receptor in the oocyte cell membrane (see, e.g., Meduri etal., J. Clin. Endocrinol. Metab. 87(5): 2266-76), and indicate that themodified superagonists of the invention may be used to improve theperformance of oocytes in patients seeking assisted reproductiontherapy.

Example 3 In Vivo Studies Using TR-4402

In vivo studies of TR4402 were performed using immature 21-day oldSprague-Dawley female rats. The FSH injection was performedsubcutaneously once a day for 3 days (0, 24 and 48 h). At 72 h bloodsamples were collected and autopsy was performed. The weight of bothovaries was measured. FSH and inhibin B levels in sera were determinedusing ICN-IRMA FSH immunoassay. Intra-ovarian estradiol content wasdetermined after homogenization of ovaries—using CT 17beta-estradiol kit(ICN Pharmaceuticals, Inc.).

An increase of ovarian weight has been previously correlated withinjected dose of FSH (Steelman and Pohley, 1953). FSH stimulatesfollicle growth (granulosa cells proliferation, hyperaemia, estradioland inhibin B production). Statistically significant differences inovarian weight (FIGS. 14A, C and D), intra-ovarian estradiol content(FIG. 15) and serum inhibin B levels (FIG. 16) after stimulation withcorresponding doses of TR-4402 and Follistim were observed. Suchadvantage of TR-4402 over wild type FSH in terms of ovarian weight,inhibin and estradiol production was observed despite a 40-50% lowerlevel of TR-4402 than Follistim remaining in sera at the end of eachexperiment (see FIG. 14B).

Since studies in rodents are generally considered as good indicators ofclinical efficacy of FSH preparations in humans, it is expected thatTR-4402 should show considerable advantage over Follistim for thetreatment of human patients. Moreover, a superactive FSH with fasterclearance rate (such as TR-4402) should have immediate applications atthe second phase of IVF protocol and result in decreased occurrence ofovarian hyperstimulation syndrome (OHSS).

Example 4 In Vitro Fertilization, Embryo Development, and Live BirthsStudies Comparing FSH Analogs to Wild Type FSH

Twenty-three day old B6D2F1 female mice (groups of 5) received onesubcutaneous injection of 10 IU of TR-4401, 10 IU TR-4901, 10 IU wildtype FSH (Follistim), or 20 IU wild type FSH (Follistim) on day one ofthe experiment. An ovulatory dose of hCG was administered by anintraperitoneal injection in at least one animal as a control.

After seventy-two hours from the FSH injection, sperm and oocytes werecollected and fertilization occurred. Sperm was collected from male B6D2and CB6F1 mice greater than 2 months of age. The male mice weresacrificed by cervical dislocation. An incision was made in the lowerarea of the abdomen, and the epididymis and vas deferens were dissectedout and placed in a sperm dish. The epididymis and vas deferens were cut3 to 5 times, and the sperm was gently squeezed out of the organs on tothe sperm dish. The sperm dish with sperm was placed in an incubator at37° C. and 5% CO2 and allowed to capacitate 30 to 90 minutes.

Oocytes were collected from the superovulated female mice which hadreceived TR-4401, TR-4901, or wild-type FSH (Follistim) by sacrificingthe female mice and dissecting out the oviducts. The oviducts wereplaced in a drops of HTF medium and the ampulae were torn to release eggclutches. The intact egg clutches were transferred to fertilizationdishes and counted. Table 2 provides the count of oocytes per group offive mice. FSH analogs TR-4401 and TR-4901 produced more oocytes at the10 IU dosage than recombinant wild type FSH (Follistem) at the 10 IU and20 IU dosages.

After the oocytes were placed in the fertilization dishes, aliquots ofsperm (1×106 to 2×106 sperm/ml) were added to each fertilization dish.The fertilization dishes were placed in the incubator at 37° C. and 5%CO2 for a minimum of four hours to allow fertilization to occur. Afterthe four hours of incubation, the fertilized eggs were transferred fromthe fertilization dishes to wash dishes where they were washed at leasttwo times in drops of 250 μl of HTF medium to remove debris. The oocyteswere stored in HTF drops in the dishes in the 37° C. and 5% CO2incubator overnight.

Twenty-four hours after fertilization, the cells were removed from theincubator. Two cell embryos were counted (Table 2, column titled “numberof 2 cell embryos”), and the fertilization rate was determined by thepercentage of oocytes which developed into two cell embryos (Table 2,column “% of 2-cell embryos”). The number of resulting two cell embryoswas greater for the groups of mice treated with FSH analogs TR-4401 andTR-4901. The fertilization rate for all groups (FSH analogs andrecombinant wild type FSH) was high.

The two-cell embryos were subsequently transferred to cultured dishesfor further development (Table 2, column titled “number of 2-cellembryos remaining in culture”) or implanted in pseudopregnant females(Table 2, column titled “number of 2-cell embryos transferred”).

The embryos which remained in the culture dishes were observed forblastocyst formation on the fourth day after fertilization. The numberof developing blastocysts is provided in Table 2 in the column titled“number of developing blastocysts”. Table 2 provides both the totalnumber of blastocysts and the number of blastocysts which hatched.

The two cell embryos which were implanted for fertilization wereimplanted in CD1 females between six and eight weeks old. Sixty 2-cellembryos were implanted in each test group of three mice with theexception of the TR-4401 group which had forty 2-cell embryos implanted.The mice were anesthetized with a solution of ketamine/zylazine byintraperitoneal injection. Once anesthetized, each mouse was shaved, anda small (0.5 cm) incision was made caudal of the rib cage and at thefirst one third of the flank of the dorsal to ventral. Another incisionwas made in the body wall to provide access to the abdominal cavity.Forceps were used to grasp the ovarian fat pad and gently withdraw theovary, oviduct, and proximal end of the uterus through the body wall.The ovary and oviduct were positioned on a cotton swab to create anangle on the ovarian-oviductal junction. The infundibulum was identifiedunder a stereomicroscope, and two pairs of superfine forceps were usedto make a hole in the bursa. Embryos were transferred by pipetting aminimal volume of M2 medium with the embryos into the infundibulum. Theorgans were then relocated into the body wall and sutured with one ortwo stitches. The skin incision was closed with one or two wound clips.The mice were observed for daily. After ten days, the recipient micewere checked for pregnancy. Table 2, column “pregnancy from 2-cellembryo transfer” provides the number of resulting pregnancies per testgroup. FSH analog TR-4901 produced the most pregnancies.

A similar experiment comparing TR-4401 to recombinant wild type FSH wasperformed using birth as an end-point. Female mice (3 mice/group) wereinjected with 1 IU hCG and 3 IU of pregnant mare serum gonadotropin(PMSG) as a control, 1 IU wild type FSH (Gonal F), 3 IU wild type FSH(Gonal F), 1 IU TR-4401, or 3 IU TR-4401. The mice were injected 48hours later with an ovulatory dose of 5 IU hCG. Twenty hours after theovulatory dose, oocytes were counted and in vitro fertilization wasallowed to take place as previously described. Subsequently, twenty2-cell embryos were implanted in pseudopregnant mothers. Table 3provides the results of this experiment. The test groups which received1 IU or 3 IU of TR-4401 achieved greater oocyte counts, higher rates ofblastocyst development, and higher birth rates compared to the controlgroup and test groups treated with recombinant wild type FSH.

TABLE 2 In vitro Fertilization and Embryo Transfer Experiment ComparingTR-4401 and TR-4901 to Recombinant Wild-Type FSH (Follistim) NumberNumber of 2-cell Pregnancy Test Article Oocytes Number % of 2-cellembryos Number from 2-cell Dose Level Count of 2-cell of 2-cell embryosremaining of developing embryo (IU/mouse) (per group) embryos embryostransferred in culture blastocysts transfer 10 IU Follistim 77 77 100% 60 (3 females) 12 6 total (3 hatched)   0 20 IU Follistim 165 161 98% 60(3 females) 101 52 (37 hatched) 2 10 IU TR-4401 207 204 99% 40 (2females) 164 75 (38 hatched) 1 10 IU TR-4901 376 369 98% 60 (3 females)171 197 (126 hatched) 3

TABLE 3 In Vitro Fertilization, Embryo Development, and Live BirthsExperiment Comparing TR-4401 to Recombinant Wild Type FSH (Gonal-F) andPMSG (control) Number of 2-cell Test Article Oocytes Number % embryosNumber % Dose Level Count of 2-cell of 2-cell remaining of developing ofdeveloping Birth (IU/mouse) (per group) embryos embryos in cultureblastocysts blastocysts Rate 3 IU PMSG 58 51 88% 31 18 58% 0/20 1 IUGonal F 26 26 100%  6 4 67% 0/20 3 IU Gonal F 21 21 100%  1 0  0% 0/20 1IU TR-4401 78 59 76% 19 12 63% 5/40 3 IU TR4401 116 113 97% 53 38 72%11/60 

Example 5 Comparison of the Quantity and Quality of Oocytes from MiceTreated with TR-4401 FSH Analog and Recombinant Wild Type FSH (Gonal F)

Oocytes from B6CBAF1 mice were quantitatively and qualitatively assessedafter in vivo treatment with a control or various doses of recombinantwild type FSH or FSH analog TR-4401 as described in Table 4. In vitrofertilization took place on day 1 (72 hours post treatment) according tothe protocol previously described.

TABLE 4 Treatment groups (3 mice/group) Treatment (Day −2) hCG (Day 0)Control: 2.5 IU PMSG (Folligon) 5 IU hCG (Chorulon) Control: hCG only 5IU hCG 0.5 IU Recombinant FSH (Gonal F) + 1 IU 5 IU hCG (Ovitrelle) hCG(Ovitrelle) 0.5 IU TR-4401 + 1 IU hCG (Ovitrelle) 5 IU hCG (Ovitrelle) 1IU Recombinant FSH (Gonal F) + 1 IU 5 IU hCG (Ovitrelle) hCG (Ovitrelle)1 IU TR-4401 + 1 IU hCG (Ovitrelle) 5 IU hCG (Ovitrelle) 3 IURecombinant FSH (Gonal F) + 1 IU 5 IU hCG (Ovitrelle) hCG (Ovitrelle) 3IU TR-4401 + 1 IU hCG (Ovitrelle) 5 IU hCG (Ovitrelle)

Treatment with TR-4401 was found to significantly increase the number ofoocytes produced. FIG. 21 provides the total number of oocytes per groupat the time of sperm washing (immediately prior to in vitrofertilization). The figure shows that TR-4401 produced more oocytes atall doses (0.5 IU, 1 IU, and 3 IU) than the test groups treated withrecombinant wild type follicle stimulating hormone.

Treatment with TR-4401 increased the total number of embryos resultingfrom in vitro fertilization. FIG. 24 provides the total number of 2-cellembryos per group. The figure shows that TR-4401 produced more 2-cellembryos at all doses (0.5 IU, 1 IU, and 3 IU) than the test groupstreated with recombinant wild type FSH.

In a similar experiment, test groups received 3 doses of 0.5, 1, or 3 IUof TR-4401 or recombinant wild type FSH (Gonal F) combined with 1 IU ofhCG. A control group received 3 doses of 1 IU hCG. On day 3, all groupswere given one ovulatory dose of 15 IU hCG. In vitro fertilization wasperformed on mice as previously described. FIG. 22 shows that micetreated with the TR-4401 FSH analog displayed higher fertilization ratesat all doses (3×(0.5, 1, 3 IU)+1 IU hCG) than mice treated withrecombinant wild type FSH (Gonal F) or the control. Furthermore, oocytesfrom mice treated with the lowest dose of TR-4401 (3×0.5 TR-4401+1 IUhCG) displayed a higher fertilization rate than those from mice treatedwith higher doses of TR-4401. FIG. 23 shows that embryos from micetreated with the TR-4401 FSH analog (all dosages) displayed higherblastocyst formation rates than the embryos from the test group treatedwith recombinant wild type FSH. Embryos from mice treated with thelowest dose of TR-4401 (3×0.5 TR-4401+1 hCG) displayed a greaterblastocyst formation rate than embryos from mice treated with higherdoses of TR-4401.

Example 6 Pharmokinetics Comparison of FSH Analogs

Pharmokinetics experiments were performed to determine the rates ofabsorption and elimination of FSH analogs TR-4401, TR-4402, and TR-4901compared to recombinant wild type FSH. A FSH Clearance Assay wasperformed to determine the amount of serum FSH in mIU/ml over time forTR-4401, TR-4402, and TR-4901 compared to recombinant wild type FSH.FIG. 17A provides the results of the assay. The figure shows delayedclearance of FSH analog TR-4401 compared to TR-4402 and TR-4901. FSHanalog TR-4402 exhibited a reduced duration of action compared to theother analogs. Similarly, FIG. 17 B shows the rate of elimination (ln[serum FSH mIU/ml] over time) for FSH analogs TR-4401, TR-4402, andTR-4901 compared to recombinant wild type FSH. TR-4402 was eliminated ata faster rate than the other analogs and recombinant wild type FSH(Follistim). Table 5 provides the data from the pharmokineticsexperiment. The data confirms that the rate of elimination (Ke) and therate of absorption (Ka) were greatest for FSH analog TR-4402. Asexpected, the serum half life (T½) was lowest for TR-4402 compared tothe other analogs and the recombinant wild type FSH.

TABLE 5 Pharmokinetics Data for FSH Analogs TR-4901, TR-4401, andTR-4402 and Recombinant Wild Type FSH (Follistem) Wild Type TR-4901TR-4401 TR-4402 FSH Ke 0.09 0.076 0.124 0.099 Ka 0.5794 0.5654 1.550.3503 V 17.47 31.55 20.46 12.51 AUC (0-t) 9328 6328 6868 8768(mIU*hour/ml) Tmax (hour) 3.8 4.1 1.8 5.0 Cmax (mIU/ml) 609.6 348.1588.6 728.8 T½ (hour) 7.7 9.1 5.6 7.0 AUC 9508.8 6570.9 6897.1 8804.5(mIU*Hour/ml)

Pharmokinetics may have a dramatic effect on how a patient reacts to aFSH analog. Hypersensitive patients at risk for hyperstimulationsyndrome may benefit from a FSH analog such as TR-4402 which acts fasterand for a shorter duration than the other analogs. Other patients wouldlikely benefit from a FSH analog such as TR-4401 which demonstrates aprolonged pharmokinetics action.

Additional pharmokinetics experiments were performed comparing TR-4401to recombinant wild type FSH (Gonal F). In one experiment mice wereinjected with a single dose of recombinant wild type FSH or TR-4401.Terminal blood levels were determined 68 hours following the injectionat necropsy. Terminal blood FSH values were at least 5-6 times higherwith TR-4401 compared to recombinant wild type FSH. Table 6 providesdosing and terminal blood FSH data.

TABLE 6 Terminal Blood FSH Data for FSH Dosage Terminal Blood FSH FSH(μg) Values (mIU/ml) Recombinant Wild Type FSH 2.22 μg all animals lessthan 2.5 (Gonal F) mIU/ml TR-4401 0.22 μg all animals less than 2.5mIU/ml TR-4401 2.2 μg all animals between 12-15 mIU/ml

Example 7 Modifications to Increase Serum Half Life of FSH Analogs

FHS analog TR-4402 was further modified by an N-terminal extension aspreviously described to enhance serum half life. Examples of furthermodifications which may enhance serum half life for FHS analogs areprovided in FIG. 18. In vitro cAMP stimulation studies using CHO cellswere conducted to compare N-terminal modified TR-4402 (LA1-4402), “wildtype” N-terminal modified FSH (LA1-Wt), TR-4402, and recombinant wildtype FSH (Follistem). FIG. 19 shows that the modifications extended theFSH serum half-life for the FSH analog TR-4402 and recombinant wild typeFHS.

An in vivo ovulation assay was conducted in hybrid B6D2F1 mice tocompare N-terminal modified TR-4402 to recombinant wild type FSH. Themice treated with N-terminal modified TR-4402 produced more oocytes thanthose treated with recombinant wild type FSH or hCG (control) for doses0.5 IU, 1.0 IU, 2.5 IU, 5.0 IU, and 10 IU. The results of the experimentare shown in FIG. 20.

All publications, patents and patent applications discussed in thisapplication are incorporated herein by reference. While in the foregoingspecification this invention has been described in relation to certainpreferred embodiments thereof, and many details have been set forth forpurposes of illustration, it will be apparent to those skilled in theart that the invention is susceptible to additional embodiments and thatcertain of the details described herein may be varied considerablywithout departing from the basic principles of the invention.

1. A modified follicle stimulating hormone (FSH), containing an aminoacid sequence which differs from the wild-type FSH, said modified FSHcomprising a modified α-subunit, wherein the potency of said modifiedFSH is increased by at least about a ten fold as compared to wild typeFSH. 2.-39. (canceled)
 40. The modified FSH of claim 1, wherein saidmodified FSH is human, wherein said α-subunit comprises at least twobasic amino acids at positions corresponding to positions 13, 14, 16,17, 20, 21, 22, 66, 68, 73, 74, and 81 of SEQ ID NO:1, and wherein saidbasic amino acids are selected from the group consisting of lysine andarginine.
 41. The modified FSH of claim 1, further comprising a modifiedβ-subunit. 42.-43. (canceled)
 44. A nucleic acid encoding the modifiedFSH α-subunit of claim
 1. 45. A vector comprising a nucleic acidencoding the modified FSH α-subunit of claim 1, wherein the vector issuitable for expressing the nucleic acid.
 46. A host cell comprising thevector of claim 45, wherein the host cell is suitable for expressing thenucleic acid. 47.-51. (canceled)
 52. The modified FSH of claim 1,wherein the plasma half-life is increased as compared to wild type FSH.53. The modified FSH of claim 52, wherein said modified FSH furthercomprises at least one sequence with a potential glycosylation siteselected from the group consisting of a sequence comprising aN-glycosylation site and a sequence comprising an O-glycosylation site.54. The modified FSH of claim 53, wherein said at least one sequencewith a potential glycosylation recognition site is an N-terminalextension on said α chain.
 55. The modified FSH of claim 54, whereinsaid N-terminal extension is selected from the group consisting of ANITV(SEQ ID No. 3) and ANITVNITV (SEQ ID No. 4). 56.-58. (canceled)
 59. Themodified FSH of claim 52, wherein said modified FSH is altered toincrease the number of negatively charged residues within the moleculeto increase plasma half-life.
 60. The method of claim 59, wherein saidnegatively charged residues are selected from the group consisting ofglutamate aspartate. 61.-66. (canceled)
 67. A method of assistingreproduction in a subject comprising administering an assisting amountof the modified FSH of claim
 1. 68. A method of diagnosing and/ortreating a condition associated with a glycoprotein hormone activity ina patient comprising administering an effective amount of the modifiedFSH of claim 1 to the patient. 69.-82. (canceled)
 83. A method ofreducing hyperstimulation syndrome in a patient undergoing assistedreproduction therapy, comprising (a) administering an assisting amountof a first modified FSH according to claim 1 wherein the plasmahalf-life of said first modified FSH is increased as compared to wildtype FSH, and (b) subsequently administering an assisting amount of asecond modified FSH according to claim 1 wherein the plasma half-life ofsaid second modified FSH is decreased as compared to said first modifiedFSH, wherein ovarian hyperstimulation is reduced as compared to when thesame patient is administered only said first modified FSH duringassisted reproduction therapy.
 84. A method of improving the quality ofoocytes in an animal comprising: administering an effective amount of asuperactive follicle stimulating hormone to said animal, wherein saidsuperactive follicle stimulating hormone contains an D-subunit with abasic amino acid at one or more positions selected from the groupconsisting of positions 13, 14, 16, and
 20. 85.-103. (canceled)
 104. Amethod of inducing superovulation in an animal comprising: administeringan effective amount of superactive follicle stimulating hormone to saidanimal, wherein said superactive follicle stimulating hormone containsan alpha-subunit with a basic amino acid at one or more positionsselected from the group consisting of positions 13, 14, 16, and
 20. 105.A method of claim 104, wherein superovulation is characterized by anincrease in oocyte number as compared to a like animal receiving thesame amount of recombinant wild type FSH. 105.-119. (canceled)
 120. Amethod of enhancing in vitro fertilization comprising: administering aneffective amount of superactive follicle stimulating hormone to saidanimal, wherein said superactive follicle stimulating hormone containsan alpha-subunit with a basic amino acid at one or more positionsselected from the group consisting of positions 13, 14, 16, and 20.120.-137. (canceled)